Review series
Lymphatic vessels and tertiary lymphoid organs
Nancy H. Ruddle
Yale University School of Public Health, New Haven, Connecticut, USA.
Tertiary lymphoid organs (TLOs) are accumulations of lymphoid cells in chronic inflammation that resemble LNs
in their cellular content and organization, high endothelial venules, and lymphatic vessels (LVs). Although acute
inflammation can result in defective LVs, TLO LVs appear to function normally in that they drain fluid and transport cells that respond to chemokines and sphingosine-1-phosphate (S1P) gradients. Molecular regulation of TLO
LVs differs from lymphangiogenesis in ontogeny with a dependence on cytokines and hematopoietic cells. Ongoing
work to elucidate the function and molecular regulation of LVs in TLOs is providing insight into therapies for conditions as diverse as lymphedema, autoimmunity, and cancer.
Introduction
Lymphatic vessels (LVs) are a series of LYVE1+/PROX1+ vessels
throughout the body that function in multiple physiologic and
pathophysiologic processes. The initial thin-walled vessels, called
capillaries, progress to collecting vessels and then to larger vessels
such as the thoracic duct. LVs also express gp38 (podoplanin),
VEGFR-2, VEGFR-3, neuropilin-2, angiopoietin-1, and CCL21,
although differences in levels of these markers exist between the
different vessels. Although other cell types can express some of
these markers, none except LVs express the entire range. PROX1
is crucial for development and maintenance of LVs (1); VEGF
receptors, especially VEGFR-3, respond to VEGF-C and VEGF-D
and induce growth of already existing LVs (2). Fluid is transported
through the LVs by means of extrinsic contraction of tissue forces
and intrinsic pumping through lymphatic muscle (3). Lymphatic
valves prevent backflow and have higher expression of PROX1 than
do the cells in the walls of the vessels.
LVs have many functions in homeostasis. They maintain fluid
balance, preventing edema by providing drainage of interstitial
fluid, provide lipid transport, and serve in an immune capacity
by carrying antigen and cells throughout the immune system and
regulating this transport through production of chemokines and
sphingosine-1-phosphate (S1P) (4). LVs are also found at sites of
chronic inflammation, referred to as ectopic or tertiary lymphoid
organs (TLOs). This Review considers how closely the functions
and regulation of LVs in TLOs resemble those throughout the
body, particularly in secondary lymphoid organs (SLOs).
SLOs
SLOs (LNs, Peyer’s patches, spleen, tonsils), which are positioned
to provide optimal interactions of components of the immune
system with invading pathogens, are remarkably organized with
T and B cell compartmentalization, APCs, stromal cells, and blood
and lymphatic endothelial cells (LECs). Cells are directed to their
various locations through the activity of chemokines produced by
several different types of stromal cells — fibroblast reticular cells,
marginal reticular cells, and endothelial cells (5). Naive cells enter
LNs through specialized blood vessels, called high endothelial venules (HEVs), and leave after interaction with antigen, which enters
into LNs via LVs. Conduits are very fine microvessels consisting of
ECM scaffolding produced by fibroblast reticular cells. They transport low-molecular-weight antigens from the cortex to the paraConflict of interest: The author has declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2014;124(3):953–959. doi:10.1172/JCI71611.
cortex and into the parenchyma of LNs, where they can contact
the HEVs (6). The organization of LNs and their vascular features
are presented in Figure 1.
The signals that organize LNs in ontogeny are tightly regulated,
which results in the development of individual LNs on a precise
temporal and anatomical schedule (7). Members of the lymphotoxin family (LTα3 and LTα1β2) play key roles in generating and
maintaining lymphoid organs (8, 9) through their production by
IL-7R+CD4+CD3– lymphoid tissue inducer cells. Lymphoid tissue
inducer cells interact with cells of mesenchymal origin, called lymphoid tissue organizer cells, which produce chemokines that in
turn induce more LTαβ. The role of stromal cells in SLO development and maintenance is becoming better understood (10) as is
the response of these cells to neuronal signals, including retinoic
acid (11). HEVs are also key organizers of LN development (12), in
that they express LTβR (13), respond to LTαβ (14, 15), and present
chemokines and adhesion molecules
Functions of LVs in SLOs
Afferent LVs bring antigen and APCs to LNs. DCs accumulate in
the subcapsular sinus and transmigrate through the floor of that
sinus into the T cell zone, while T cells access the parenchyma of
the LN through the peripheral medullary sinuses (16). Efferent
LVs drain activated cells from the LN, which then travel through
afferent vessels to the next LN in the series, into the thoracic duct,
and into the bloodstream via the subclavian veins. Markers that
distinguish efferent from afferent vessels are not yet known.
Cellular trafficking between the periphery and within LNs is regulated by the lymphoid chemokines CCL19, CCL21, and CXCL13
and by S1P (17). CCL21 is produced or expressed in LNs by stromal
cells, HEVs, and LECs. CCR7, the ligand for CCL19 and CCL21, is
found on DCs and naive and memory T cells in the blood and is
downregulated as they enter into the parenchyma of the LNs. S1P
is found in high concentrations in the blood and lymph, where
lymphocytes express low levels of the receptor, S1PR1. The receptor
is re-expressed at higher levels in the LN, where S1P levels are low.
Egress of S1P1R-expressing activated and memory cells from LNs is
regulated by the high concentration of S1P in the lymph (ref. 17 and
summarized in ref. 18). Thus, LVs play crucial roles in trafficking in
LNs via activation and the expression of chemokines and S1P.
Additional potential functions of LVs in LNs include a role in
tolerance, as they express self-antigen under the control of AIRE
for presentation to T cells (19). LVs may also serve as organizers of
LNs in the same manner as HEVs. Conditional inducible knockouts regulated by PROX1 will provide insight into this question.
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Figure 1
Comparison of a LN and a salivary gland TLO. Note the presence in both of T and B cell organization, APCs, stromal cells, conduits, chemokines,
HEVs, and LVs. One difference is the apparent absence of a capsule in the TLO compared with the LN. Another possible difference concerns
uncertainty regarding whether the TLO LVs are afferent and/or efferent. FDC, follicular DC.
TLOs
TLOs, also referred to as ectopic lymphoid tissues, are accumulations of cells in chronic inflammation (reviewed in ref. 20) and are
referred to as tertiary to distinguish them from SLOs. SLOs arise
during development at key locations in the body under the control
of a precise developmental program, but chronic immune activity
in the adult can give rise to similarly organized accumulations of
lymphoid cells in almost any non-lymphoid tissue through a process that is not preprogrammed but rather the result of induction
by factors in the environment that could elicit the same signals
that contribute to LN development (reviewed in refs. 20, 21). TLOs
are characterized by their cellular, organizational, chemokine, and
vascular similarity to SLOs, especially LNs. These similarities
include T and B cell compartmentalization, APCs such as DCs and
follicular DCs, stromal cells (5), conduits, and a highly organized
vascular system of HEVs and LVs (see below) (5, 22, 23). Common
features of TLOs and LNs are depicted in Figure 1.
It has been suggested that TLOs differ from SLOs by the absence
of a capsule; however, TLOs in a variety of chronic kidney diseases
are in contact with a fibrous capsule (24). The absence of a capsule may reflect the manner in which TLOs arise, compared with
LNs. As noted above, the latter arise at predefined locations as a
result of stromal and endothelial organizers. Even though most of
these elements are also present in TLOs (5), the order in which they
populate the TLO may differ from the precise temporal aspect of
LNs. The general absence of a capsule may have consequences for
trafficking patterns that could differ in TLOs from what is seen in
LNs, in which the DCs and T cells migrate through the peripheral
medullary sinus to the parenchyma (16).
TLOs arise in several instances of chronic inflammation,
including autoimmunity, chronic graft rejection, persistent
infection (summarized in ref. 20), atherosclerosis (25), and
cancer (26–28). They can be induced experimentally by tissue954
specific expression of certain inflammatory mediators (summarized in ref. 20), including members of the lymphotoxin family
(15, 29), the very cytokines that are crucial for lymphoid organ
development and maintenance (8).
As noted above, lymphoid chemokines regulate trafficking of
lymphocytes and DCs to and within LNs, and their expression
(20, 30) is one criterion that defines TLOs. Chemokines play similar roles in LNs and ectopic lymphoid accumulations, since the
CCR7-expressing T cells and DCs are situated in the same locality
as CCL19 and CCL21 and the CXCR5+ B cells are in regions with
high CXCL13 levels. TLOs likely function as local sites of antigen presentation and lymphocyte activation, including somatic
hypermutation and class switching in B cells (31), which suggests
that they facilitate local antimicrobial responses as well as epitope
spreading (32, 33) and autoimmune exacerbation.
TLOs can progress from a relatively benign to a destructive
phase and lose their lymphoid organ characteristics. For example,
in the non-obese diabetic (NOD) mouse model of type 1 diabetes
mellitus (T1DM), initial pancreatic infiltrates are characterized
by HEV development and minimal islet destruction, whereas later
stages demonstrate frank islet destruction and diabetes (34). In
this model, pancreatic TLOs disappear after removal of the β cell
antigen and are replaced by tissue fibroblasts. The presence of high
proportions of regulatory T cells in some TLOs (25, 35) suggests
that immune regulation occurs in these locations.
Functions of LVs in TLOs
Thin-walled vessels with typical lymphatic markers, including
LYVE1 and PROX1, are apparent in the TLOs arising in both clinical settings (autoimmune disease and chronic graft rejection) and
experimental models (summarized in ref. 20) (23, 25, 36–40). They
are also found in TLOs associated with some tumors (35). The functions of the LVs in TLOs have not been thoroughly investigated but
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are worthy of analysis with regard to fluid balance and transport of
antigen and naive, activated, and memory lymphocytes.
Fluid balance is a critical function of LVs in the body. Although
production of IL-7 and IL-7Rα by afferent LVs is crucial with
regard to fluid drainage in the skin during acute inflammation
(41), this has not been investigated with regard to the chronic
inflammation of TLOs. Since edema is a frequent occurrence in
acute inflammation, it is likely that LVs associated with that process serve a drainage function. Lymphangiogenesis accompanies
acute inflammation with enhanced lymph flow (42). Seeger and
colleagues suggest that inflammation occurs before lymphangiogenesis and gives rise to that process (43). Lymphangiogenesis at
early times after immunization or during acute inflammation
may be the result of the presence of excess fluid, but the LVs are
unable to transport APCs (13) due to defects in lymphatic contraction (44). Thaunat and colleagues have suggested that the
existence of edema in an injured tissue results in an insufficient
lymphatic outflow that then gives rise to chronic inflammation at
the local site (45). They suggest that defective lymphatic drainage
is a prerequisite for the development of TLOs and have provided
data supporting this concept in chronic graft rejection. Once the
TLO has developed, the LVs may function normally and provide
fluid balance. Techniques to measure fluid accumulation are
available (46) but have not been employed in the local region of
a TLO. Future research could take advantage of measurements of
interstitial fluid pressure in the local vicinity of a TLO to evaluate
whether edema occurs and the LVs are functional. This may be
more feasible and meaningful in a clinical setting, in which a relatively accessible TLO such as the joint in rheumatoid arthritis (RA)
provides a logical study site.
Do TLO LVs transport soluble or cell-associated antigen as they
do in LNs? Perhaps this is a moot point, because in contrast to
SLOs, the antigen is continuously locally available in TLOs. For
example, proteins such as insulin in the pancreatic islet are in
immediate proximity to or even a part of the TLO in T1DM. Thus,
the necessity for antigen transport to the TLO through an extensive LV system might not be necessary, and the LVs may not serve
that function. The presentation of self-antigen in LNs (19) that
has been suggested as a mechanism for self-tolerance has not been
investigated in TLOs and is an important area for future research.
LVs transport cells to primary LNs and then to downstream LNs.
Is there such a cellular transport function for TLO-associated LVs,
in which cells travel to and from the LNs? LVs that are packed with
lymphocytes are prominent in some TLOs (ref. 36 and Figure 2),
which suggests that they do transport lymphocytes. Additional
evidence supporting an afferent function for TLO LVs comes
from the study of myasthenia gravis (MG), an autoimmune disease
characterized by thymic TLOs with HEVs and B cell follicles. HEVs
are the normal entry points for lymphocytes from the circulation
into the LN parenchyma, but in this particular case, the HEVs are
negative for CCL21, suggesting that it could be difficult for CCR7+
cells to enter through their usual (HEV) route. In most humans,
thymic LVs do not normally express CCL21, but in patients with
MG, thymic PROX1+VEGFR-3+ vessels do express this chemokine
(47). Because B and T cells in this instance express CCR7 and naive
B cells migrate across a CCL21 gradient, the authors suggest that
although the thymus does not normally contain afferent LVs, in
this pathologic TLO, the LVs function in that capacity and contribute to thymic hyperplasia. In the early stages of non-obese diabetes, the infiltrates exhibit the characteristics of TLOs, includ
ing HEVs and LVs (23, 48, 49), T and B cells, and macrophages.
CCL21 is expressed on such LVs and is reduced upon treatment
with complete Freund’s adjuvant (49), as is the lymphocytic infiltration, which again suggests that the CCL21-expressing LVs act
as afferent vessels into the TLOs.
TLO-associated LVs may also function in an efferent capacity
by transporting activated lymphocytes to the periphery. CCL21
is a crucial chemokine for the traffic of LV-resident DCs to LNs;
therefore, the presence of CCL21+ LVs in TLOs would support
the idea that they can serve to transport CCR7 + DCs and lymphocytes. Indeed, there are several examples of CCL21 expression by TLO-associated LVs in humans with RA, Crohn’s disease,
Sjögren’s syndrome, lichen planus (50), and chronic allograft
rejection (51) and in the TLOs of pancreatic infiltrates in NOD
(23) and RIPLTα mice (30).
Support for the idea that LVs in TLOs serve an efferent function comes from the observation that continual administration
of FTY720, an S1P receptor agonist that blocks lymphocyte egress
from LNs (52), prevents diabetes in NOD mice (53). This is only
effective if the mice have already developed pancreatic TLOs (23).
Treatment results in additional accumulation of lymphocytes in
the pancreatic TLOs, which is reversed upon cessation, resulting in
rapid islet destruction and diabetes (23, 54). These data suggest that
lymphocyte trafficking through LVs in TLOs in NOD mice occurs
under the control of the lymph S1P gradient and re-expression of
its receptor by T cells, as occurs in LNs. These data are also consistent with the observation that FTY720 also prevents egress from
inflamed tissues into afferent lymphatics (55, 56) and raise the exciting possibility that inhibitors of LV function could prevent diabetes
and other autoimmune diseases systemically by preventing trafficking from the TLO to the LNs. It is not known whether the LVs in
TLOs produce S1P as they do in the rest of the body (4).
Regulation of LVs in TLOs
During ontogeny, LVs develop after the embryonic blood vessels
have formed, sprouting from the cardinal vein (57). These events
are orchestrated by products of the homeobox genes (SOX18 and
PROX1) and growth factors and their receptors (VEGF-C and
VEGFR-3) and require platelets (ref. 58 and reviewed in ref. 59). The
involvement of additional cell types is suggested by the existence of
lymphangioblasts, which are distinct from blood endothelial cells,
in developing tadpoles (60). Studies in chickens (61, 62) and mice
(63–65) support a role for mesodermal cells that express macrophage and lymphatic markers that become, integrate into, or support LVs through their production of VEGFs.
Regulation of lymphangiogenesis in TLOs is poorly understood.
It is not known whether LVs precede the cellular accumulations
and organization into TLOs. Since LVs produce the lymphoid
chemokine CCL21, it is possible that they are among the stromal
organizers of LNs and TLOs. This is consistent with the role that
HEVs play in LNs (12) and presumably in TLOs, since HEVs are
similarly, if not identically, regulated in those sites (15). Data from
doxycycline-induced TLOs in RIPLTα mice indicate that the presence of LVs is a very early event that can occur prior to apparent
leukocytic infiltration into the affected organ (37) and that occurs
in the absence of obvious edema. Angiogenesis occurs in inflammation and platelets are present, which indicates that the important players in embryonic lymphangiogenesis may participate.
Thus, the possibility, though remote, exists that a recapitulation
of the developmental program could occur — that is, that LVs in
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Figure 2
Diagrammatic rendering of actual staining of a TLO from a mouse salivary gland. (A) Giemsa staining of TLO reveals the presence of leukocytes. (B) Staining for B cells (B220, green) and T cells (CD3, red).
(C) Staining for HEVs (MECA 79, red) and LVs (LYVE1, green). Note
that the LVs are filled with lymphocytes, which indicates that transport
of these cells is occurring and, thus, suggests that these LVs possess
afferent and/or efferent function. Adapted from Proceedings of the
National Academy of Sciences of the United States of America (36);
copyright (2007) National Academy of Sciences, USA.
inflammation could arise from veins as they do in development.
The presence of vessels that express markers of HEVs and LVs in
the inflamed LN supports this scenario (13). On the other hand,
the presence of blood vessels and their nearby LVs in TLOs suggests that lymphangiogenesis in inflammation occurs by sprouting from existing LVs. But what are the cells that signal these
events? DCs, macrophages, and T and B cells have been implicated
956
in the regulation of LVs in acute inflammation (13, 66–68). More
recently, neutrophils have also been proposed to increase bioavailability of VEGF-A and to produce VEGF-D (69). Different cells
may be important at different times in various tissues. For example, B cells appear to be important in the lymphangiogenesis that
occurs in LNs during inflammation, but only at the early stages
after immunization (13, 69).
The participation of macrophages in lymphangiogenesis in
acute inflammation has been well documented, although the precise nature of their role is a subject of considerable controversy.
Data in a model of corneal transplant lymphangiogenesis suggest that macrophages can actually transdifferentiate into LECs
(70); that is, that macrophages themselves are precursors to LECs
(61, 62, 64, 71). The expression of LYVE1 by macrophages could be
interpreted as evidence that this is the case. On the other hand, the
expression of this marker on both cell types may be a red herring.
The presence of additional LV markers such as PROX1 would go
some way toward resolving this issue.
The well-known capacity of macrophages to produce growth
factors, such as VEGF-C, VEGF-A, VEGF-D, PDGF, and adrenomedullin (summarized in ref. 72) suggests that it is likely that
these cells contribute indirectly. Macrophages produce TNF-α,
which has been implicated in lymphangiogenesis in a model of
acute Mycoplasma pulmonis lung infection (73), which also supports
an indirect mechanism for the functions of macrophages in LVs
rather than direct transdifferentiation. Kerjaschki and colleagues
demonstrated the presence of host bone marrow–derived precursors in association with LVs in the TLOs of chronically rejecting
kidneys (39). Osteoclast precursors, which include cells with macrophage properties, participate in lymphangiogenesis in a model
of TNF transgene– and serum-mediated RA (74). During low-dose,
streptozotocin-induced pancreatic inflammation, there is a marked
increase in macrophages in and around the islets. One population
produces VEGF-C, and another LYVE1+ population appears to
integrate or differentiate into LECs (75). As in ontogeny or acute
inflammation, it is unclear whether macrophages differentiate into
LECs, integrate into LVs, and/or produce growth factors in TLOs.
Lineage-tracing experiments might resolve this controversy.
Cytokines contribute to lymphangiogenesis in acute inflammation, although there have been few studies evaluating their roles in
the chronic inflammation–associated TLOs. The question of LT
participation is of particular interest given both its crucial role in
lymphoid organ development and its ability to induce TLOs. LTα
appears to play a minor, but real, role in lymphangiogenesis in
development, as Lta–/– mice exhibit somewhat diminished LV function (37). Both LTα and TNF-α contribute to lymphangiogenesis
in acute inflammation in M. pulmonis infection (37, 73). LTα also
participates in acute lymphangiogenesis in the skin (37). LVs are
induced in TLOs of RIPLTα mice. In this case, lymphangiogenesis
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occurs in the absence of LTβ (37). On the other hand, in a model
of CXCL13-induced thyroid TLO, LVs are inhibited by treatment
with LTβR-Ig (38), implicating the LTαβ complex. Further experimentation in spontaneous TLOs, such as Sjögren’s syndrome, RA,
and T1DM may reveal which cytokines, in addition to the TNF
family, contribute to this process, whether the classical or alternative NF-κB pathways are involved, and whether these effects
directly modulate LVs and/or are mediated indirectly through
their induction of infiltrating cells that produce growth factors
such as VEGF-C or VEGF-D.
The plasticity of LVs is a reflection of their environment, which
influences their function, especially in the case of inflammation.
CCL21 is particularly sensitive to inflammation. The low level of
CCL21 protein on mouse dermal LVs is increased in explanted
TNF-α–treated skin or oxazolone-induced contact sensitivity
(76). TNF-α treatment of human dermal LECs results in increased
CCL21 mRNA transcription, translation, and cell surface protein expression (76). As noted above, CCL21 expression becomes
detectable on LVs in the thymus of TLOs in patients with MG (47).
Immunofluorescence and microarray studies that compare LECs
from acutely inflamed and resting mouse skin reveal increased
expression of CCL21 and several other inflammatory genes (77).
Interestingly, there is downregulation of other genes, including
Vegfr3 and Prox1. Extension of these studies to LVs in TLOs may
reveal differences due to the chronic nature of stimulation, and
data from a mouse corneal model of recurrent inflammation suggest that this is the case (78). The authors suggest that, in chronic
inflammation, LVs retain memory in their accelerated development of a network of functional LVs (78). The ability to isolate LVs
on the basis of their antigen expression (77) or transgene induced
fluorescence (79) will allow their molecular analysis and comparison to vessels from resting LNs, activated LNs, and TLOs.
Future research directions
The functions of LVs in TLOs may be revealed by a detailed molecular signature. Are there are differences in gene expression between
afferent and efferent LVs in LNs, and is this reflected in different
LVs in TLOs? As noted above, new techniques allowing isolation,
purification, and single-cell in situ analysis will provide the tools
for determination of gene expression. Are there any genes that are
differentially expressed in LVs from TLOs compared with those
in the rest of the body? If so, it will be possible to preferentially
affect those vessels with inhibitors of their function while leaving
remaining LVs intact.
Analysis of lymphocyte, DC, and antigen-trafficking patterns
in TLOs in real time in vivo is now possible with the use of mice
that express fluorescent markers for HEVs (80) and LVs (79, 81).
Although the technique of in vivo imaging is well established for
analysis of trafficking in LNs (82), addressing this issue in TLOs
is a greater challenge. The key is to study TLOs in tissues that
are amenable to these techniques, which will allow the evaluation
of questions concerning LV insufficiency, memory, and plasticity
in LVs in TLOs, and on a functional level, to determine whether
valves and muscles occur in these vessels. The analysis of TLOs
in mice whose LVs are conditionally deleted or induced through
PROX1 regulation will provide information regarding the importance of fluid drainage functions of LVs in this context. Finally,
the influence of the local environment on the LVs in TLOs in
different organs must be analyzed. How do differences the local
milieu of the pancreas compared with that of the digestive tract,
salivary gland, or joint influence the LVs that arise in the TLOs
in those organs during T1DM, inflammatory bowel disease,
Sjögren’s syndrome, or RA?
Clinical implications
Elucidation of LV regulatory mechanisms in TLOs will contribute
to the development of therapies to either promote or inhibit their
formation, but we must keep in mind that LVs may be beneficial
or detrimental depending on their context. The following sections
discuss the implications of LV modulation in autoimmunity,
cancer, and fluid balance/lymphedema.
Autoimmunity. Inhibition of TLO LVs may be beneficial in autoimmunity, since they contribute to exacerbation by epitope spreading; this could occur through their transport of naive lymphocytes
and APCs to the local site. Treatments that discourage LV development, such as inhibition of macrophages (clodronate), cytokines
(antibodies or receptor blockers), and growth factors (e.g., VEGFs)
or those agents that inhibit function, such as the S1P1 receptor
agonist FTY720, could be beneficial. The identification of markers preferentially expressed or expressed at a high level on TLO
LVs would allow for their preferential inhibition. Inhibition of LV
function might be protective in MG. LVs most likely contribute
to priming by bringing naive lymphocytes into the thymic TLOs,
thus leading to sensitization to the nicotinic AChR and pathogenic antibodies to that antigen.
One must be cautious about the possibility that wide-scale
inhibition of LVs might result in disastrous lymphedema. Thus,
the goal should be targeted delivery to the TLO by vehicles
such as nanoparticles (83, 84) that could deliver LV inhibitors
(e.g., VEGFR-3–targeted antibodies) at the selected site. One can
envision induced temporary perturbations of LV function while
leaving the main beneficial functions intact. This will be possible
as more information becomes available regarding the special properties and regulation of LVs in TLOs.
Cancer. Lymphangiogenesis occurs in many human tumors
(summarized in ref. 85), and an enormous literature emphasizes a detrimental role for LVs as contributors to metastasis. A
logical approach to inhibiting this spread would be the use of
lymphangiogenesis inhibitors (85). A summary of the actual
agents that are in clinical use for this purpose is presented in a
recent review article (86). As noted above, the hazards of such
approaches can lead to unintended lymphedema.
It may be time to rethink the approach of inhibiting LVs in cancer, as they may contribute to defense in some cancers. TLOs are
clearly beneficial in some cancers, particularly if there is a generous component of HEVs (28). The interpretation of these data is
that the TLO serves as a site of priming of naïve cells that enter
via the HEVs and become sensitized to the tumor antigens. Since
the high number of HEVs also correlates with reduced metastases,
we need to consider that the response generated in the tumor is
effective elsewhere. Perhaps this occurs because memory cells leave
the tumor TLO via LVs, and the activated cells traffic throughout
the body and seek out metastatic disease. Thus, in this context,
the LVs in tumor TLOs are beneficial, as they serve as an exit for
activated lymphocytes. It might be of benefit to encourage the
development of LVs in this context (28).
Lymphedema. Engineering artificial TLOs may be of benefit in
counteracting the effects of lymphedema. LVs in TLOs most likely
contribute to fluid balance; understanding the roles of cytokines
and cells in LVs in TLOs could be advantageous in efforts to
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encourage lymphangiogenesis and fluid drainage in situations
where the LVs are defective. It has been estimated that 140–250
million people worldwide suffer from this painful, disfiguring
problem, which can be the result of an anatomical abnormality
of LVs of genetic origin (primary lymphedema) or the result of
damage or obstruction of LVs, due to cancer, cancer treatment
(surgery or radiation), or infection (secondary lymphedema). The
use of inflammation-induced LVs might be beneficial in these
situations. However, as noted above, the lymphangiogenesis that
initially occurs in acute inflammation can result in defective LVs
(44); therefore, it is crucial to understand the factors that generate
and maintain functional, mature, LVs.
concerning LVs in general and in TLOs in particular. The presence of LVs in TLOs has been well documented, and evidence for
their functions has been explored. Methods, particularly imaging techniques, to further probe the functions of LVs in LNs and
TLOs are available and continually improving. The regulation of
lymphangiogenesis in TLOs remains a fertile area of research and
is of key importance for the prophylaxis and treatment of autoimmunity, cancer, and lymphedema.
Conclusions
The secrets of LVs are being rapidly revealed, but there is much
to be learned. Their existence in TLOs adds to the complexity
and excitement in this emerging field. Major questions remain
Address correspondence to: Nancy H. Ruddle, Department of Epidemiology of Microbial Diseases, Yale University School of Public
Health, 60 College St., New Haven, Connecticut 06510, USA. Phone:
203.785.3281; Fax: 203.737.2921; E-mail: nancy.ruddle@yale.edu.
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Acknowledgments
Work in the author’s laboratory was supported by NIH grants
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